The p53 gene is mutated in over 50 different types of human cancers, including familial and spontaneous cancers, and is believed to be the most commonly mutated gene in human cancer (Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al., Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of mutations in the p53 gene are missense mutations that alter a single amino acid that inactivates p53 function. Aberrant forms of human p53 are associated with poor prognosis, more aggressive tumors, metastasis, and short survival rates (Mitsudomi et al., Clin Cancer Res 2000 October; 6(10):4055-63; Koshland, Science (1993) 262:1953).
The human p53 protein normally functions as a central integrator of signals including DNA damage, hypoxia, nucleotide deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8). In response to these signals, p53 protein levels are greatly increased with the result that the accumulated p53 activates cell cycle arrest or apoptosis depending on the nature and strength of these signals. Indeed, multiple lines of experimental evidence have pointed to a key role for p53 as a tumor suppressor (Levine, Cell (1997) 88:323-331). For example, homozygous p53 “knockout” mice are developmentally normal but exhibit nearly 100% incidence of neoplasia in the first year of life (Donehower et al., Nature (1992) 356:215-221).
The biochemical mechanisms and pathways through which p53 functions in normal and cancerous cells are not fully understood, but one clearly important aspect of p53 function is its activity as a gene-specific transcriptional activator. Among the genes with known p53-response elements are several with well-characterized roles in either regulation of the cell cycle or apoptosis, including GADD45, p21Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2 (Levine, Cell (1997) 88:323-331).
Mitogen-activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs) are important proline-directed Ser/Thr kinases that play critical roles in cell differentiation and proliferation. PRP (pre-mRNA processing gene) is a CDK-like kinase with homology to MAPKs (Huang, Y. et al. (2000) Biochem Biophys Res Commun; 271(2): 456-63).
Pre-mRNA processing 4 (PRP4) is a nuclear serine-threonine kinase activated by EGF stimulation, plays a role in transcriptional regulation, and may be involved in pre-mRNA splicing and intracellular signaling (Kojima, T. et al. (2001) J Biol Chem 276, 32247-56; Gross, T. et al. (1997) Nucleic Acids Res. 25: 1028-1035). The Prp4 gene of Schizosaccharomyces pombe encodes a protein kinase that appears to be involved in pre-mRNA splicing (Gross et al., supra). The sequence of PRP4 kinase and its function in pre-mRNA splicing are highly conserved in yeast and humans (Wang, et al., Hum Mol Genet. (1997) November; 6(12):2117-26; Schwelnus et al., EMBO Rep. (2001) January; 2(1):35-41). Based on kinase domain sequence, Prp4 belongs to the Clk (CDC-like kinase) family and interacts with CLK1 (Kojima et al., supra).
The ability to manipulate the genomes of model organisms such as C. elegans provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Dulubova I, et al, J Neurochem 2001 April; 77(1):229-38; Cai T, et al., Diabetologia 2001 January; 44(1):81-8; Pasquinelli A E, et al., Nature. 2000 Nov. 2; 408(6808):37-8; Ivanov I P, et al., EMBO J 2000 Apr. 17; 19(8):1907-17; Vajo Z et al., Mamm Genome 1999 October; 10(10):1000-4). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as p53, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
All references cited herein, including sequence information in referenced Genbank identifier numbers and website references, are incorporated herein in their entireties.